Use of multiplex PCR in pleural effusion: is it necessary to change the paradigm of culture-based methods?

The microbiological diagnosis of pleural effusion is based largely on classical microbiology methods, but these methods have a high rate of false negative results. Some previous studies have shown improved diagnostic performance for pathogens such as Streptococcus pneumoniae using molecular biology methods. We present the use of a multiplex PCR platform (BIOFIRE FILMARRAY Pneumonia Panel) for the aetiological diagnosis of pleural effusion in paediatric pneumonia. We present a case series of 17 pleural fluid samples that were processed by culture-based microbiology and molecular biology methods. Microbiological isolation was successful in four cases (25 %) through traditional culture methods. In contrast, the molecular biology panels allowed for detection in 16 out of 17 cases (94 %). The results from these panels led to a change in management for nine out of the 17 cases (52 %). This study found an increase in aetiological diagnosis in complicated pneumonia in children by using molecular biology methods, which led to a significant change in patient management.


INTRODUCTION
The microbiological diagnosis of pleural effusion (PE) is based largely on methods of classical microbiology.Culture-based methods allow for testing of bacterial susceptibility, but these methods take time and frequently have false negative results related to previous antimicrobial administration, among others [1].Some studies have reported an improvement in microbiological diagnosis, especially for Streptococcus pneumoniae, when using PCR and culture methods versus only culture methods [2].In a case series of adults, the multiplex PCR assay identified pathogens more frequently than conventional methods [23.3 % (7/30) vs. 6.7 % (2/30), P=0.008] [3].A paediatric case series using the BioFire Film Array Blood Culture Identification (BCID) Panel (BioFire Diagnostics) on pleural fluid samples, presented as Meeting Abstracts in 2020, revealed that 10 samples were positive with the BCID panel (eight for Streptococcus pneumoniae, one for Staphylococcus aureus and one for Streptococcus pyogenes), compared to only 2/10 positive cultures [4].Other studies have used the BCID panel in different sterile body fluids, including PE, although there are currently no large studies that support its use in these fluids [5,6].
In Colombia, recent reports have documented an increase in complications related to Streptococcus pneumoniae pneumonia due to serotype replacement [7] and circulation of more aggressive adenoviruses in children (including more pleural effusion cases) [8].According to the literature, Pneumococcus has been reported more frequently in samples sent for PCR than in samples sent for culture [2,9].Adenovirus cannot be identified in pleural fluid using conventional microbiology techniques.Therefore, molecular biology techniques are important to improve aetiological diagnosis for these samples.
Here we describe the utility of multiplex PCR in pleural fluid for the diagnosis of complicated pneumonia in children by using a commercial platform for respiratory samples.We chose to utilize this platform because it is the most commonly used in the country and is the primary one employed by most hospitals with molecular biology capabilities.

Study population
We conducted a retrospective, descriptive, case series of children with pneumonia complicated by PE, empyema or necrotizing pneumonia who underwent surgery.Pleural fluid was sent to the laboratory for culture and cytochemical testing analysis between 2021 and 2022 in a reference children's hospital in Bogotá, Colombia.The paediatric infectious diseases (ID) and paediatric intensive care unit (PICU) team determined in which cases to use the surplus of pleural fluid to test multiplex PCR.

Microbiology and multiplex PCR
All pleural fluid samples were analysed for conventional cultures and cytochemical testing.We cultured samples in sheep blood agar, chocolate agar and MacConkey agar (bioMérieux).We reported growth every 24 h for at least 4 days (all cultures were taken with sterile techniques during surgery).If any growth was detected, the colony was identified and tested for antimicrobial resistance using the BD Phoenix 100 system (Becton Dickinson).We used the BIOFIRE FILMARRAY Pneumonia Panel (BioFire Diagnostics).The panel is a multiplex PCR device with 27 common lower respiratory tract infection pathogens approved by the FDA for use in sputum (including endotracheal aspirate) and bronchoalveolar lavage (including mini-BAL) sample types.The panel includes 18 bacterial pathogens, nine viral pathogens and seven antibiotic resistance genes.We took 200 µl of pleural fluid and processed the sample following the same manufacturer's recommendations for respiratory samples.All samples were processed promptly upon arrival at the laboratory, at room temperature, within 1 h of their arrival.The results were analysed by a multidisciplinary team that included: two paediatric infectious disease physicians, paediatric critical care physicians and microbiology experts.Any change in a patient's therapeutics was decided by this group.

Statistical methods
We used R version 4.2.2(The R Foundation for Statistical Computing Platform) to analyse and tabulate the information.We describe all variables in this study using descriptive statistics.

RESULTS
We identified 17 samples that were processed for multiplex PCR from 16 patients (Table 1).One patient was taken twice for surgery and sample collection due to two PE events.The median age was 3 years (interquartile range 2-4 years) and 5/16 (31.2 %) were female patients.Fever and respiratory distress were common signs in 14 of 16 patients (87.5 %).Pleural empyema was the most common presentation (7/17, 41 %), followed by free PE (6/17, 35 %) and necrotizing pneumonia with empyema (4/17, 23.5 %).We found viral infection, prior to the appearance of a complicated pneumonia episode, in nine of 16 patients (52.8 %), adenovirus in three cases, rhinovirus/enterovirus in two, parainfluenza virus 3 in two, and parainfluenza 2 and 4 in one.All samples were collected by videoassisted thoracoscopic surgery (VATS).Blood and pleural fluid cultures were done for all patients.
Pathogens were documented in 16 out of 17 (94 %) molecular panels performed.Streptococcus pneumoniae was detected in 11 out of 17 (64.7 %) multiplex PCR tests, adenovirus was detected in seven out of 17 (41.1 %) and Haemophilus influenzae in two out of 17 (11.7 %) tests.Other pathogens were detected in one test each, such as rhinovirus/enterovirus, human metapneumovirus, Enterobacter cloacae and Pseudomonas aeruginosa.No resistance genes were identified by PCR.Regarding pleural fluid and blood cultures, they were positive in four (25 %) patients (three patients with positive blood cultures and two with pleural fluid isolation).Streptococcus pneumoniae was isolated in four patients (23.5 %), of which only one was in pleural fluid.The results of semiquantitative PCR were assessed in each case.Nonetheless, given the sterility of the liquid, any detection was regarded as significant.
We found that the use of multiple PCR allowed for some changes in antibiotic management in nine out of 17 (52.9%) of the patients.In five patients there was an increase in the antibiotic spectrum, and in four patients there was a decrease in the antibiotic spectrum.

DISCUSSION
The performance of culture-based microbiology tests for the aetiological diagnosis of pneumonia with PE is low [1,9].In paediatrics, Streptococcus pneumoniae and viruses are a very important cause of PE, the latter being mainly related to small and less-symptomatic effusions.Other bacteria such as Staphylococcus aureus, Streptococcus pyogenes and H. influenzae are also important causes of PE.Previous studies have documented that the recovery of Streptococcus pneumoniae in culture is difficult; it has been reported with 24 % positivity in conventional cultures compared to 71 % with PCR-based techniques [2].This case series found an increase in pathogen detection (23.5 % in cultures vs. 94 % in multiplex PCR tests).We also documented three times more diagnoses of pneumococcus using PCR than using cultures in complicated pneumonia with PE.
Previous studies have shown that 31 % of patients with adenovirus pneumonia can be complicated by PE, this being much more frequent in patients with a severe presentation (up to 79 %) [10].This study was carried out during a peak of severe adenoviral infections in Colombia [8].We detected adenovirus in 41 % of the samples, of which in almost half no related bacterial pathogen was documented.In addition, all these patients with adenoviral infection without documented coinfection presented as free PE.This study also documented detection of virus in 52 % of PE samples, emphasizing the importance of these viruses in some cases of complicated pneumonia in paediatrics.A case-by-case analysis is required to determine if there are pathogens related to the PE beyond possible bacterial coinfections.In most detections of adenovirus in PE without detection of other copathogens (with PCR and culture techniques, and no other focus that could explain the presence of Staphylococcus aureus), clindamycin was stopped and although ceftriaxone was maintained, short courses of treatment were used in selected cases according to ID and PICU team analysis.
This case series indicates the importance of implementing PCR-based technologies for the diagnosis of PE in paediatrics and its implications as part of an Antimicrobial Stewardship Programme.Similar to previous studies [1,2], it describes the usefulness for the diagnosis of PE especially due to Pneumococcus, the most frequent cause of complicated pneumonia in children.Interestingly, it also improved virus identification previously not possible with available tests to the clinicians.It is essential to analyse the results in relation to the clinical situation of the patient, as there could be false positives related to contamination.A standardized protocol that avoids sample contamination with other unrelated micro-organisms is necessary.Additional robust studies are needed to validate the use of existing platforms on the market, such as the one used in this study.For this initial descriptive study, we did not employ positive or negative controls for the panels used.Nevertheless, all samples that tested positive for Streptococcus pneumoniae in conventional culture also tested positive in the multiplex PCR.In the case of patient 1, who had no growth detected in the culture, no detection was observed in the multiplex PCR either.However, it is our hope that in a much larger study, much stricter controls can be implemented.
Although the use of this multiplex PCR device is not approved by the FDA for use in pleural fluid samples and its use in PE is considered off-label, this case series describes its potential usefulness and importance in contexts where there is no other approved PCR technology for diagnosis in pleural fluid samples.This could enhance the prospects of achieving a timely diagnosis and improving antibiotic therapy for paediatric patients with complicated pneumonia The authors decided to use the BIOFIRE® FILMARRAY® Pneumonia Panel following manufacturer's recommendations.However, as they state in line 166 this platform is not FDA approved for use in pleural fluid samples.Why did the authors select this platform then?Are others more suitable for the clinical samples interrogated?2. If this platform is not approved, the authors need to explain how they controlled the pcr samples.What positive and negative controls were used?How can they identify false positive/negative results in coinfections and in single species infection?How where the samples stored prior to analyses?These details should be included in the materials and methods and discussed as it is known that storage conditions of PE samples can affect PCR results.Minor points: 1. Have the authors, following the kit's recommendation, performed semi-quantitative analysis for the most common bacteria identified?Did they check for presence of resistance genes? 2. The authors should discuss the impact of anaerobes (if any) in pediatric pneumonia as has been the case in adults.3. Reference missing in line 50.1. Methodological rigour, reproducibility and availability of underlying data Reproducibility will be very difficult with these specific clinical samples.2. Presentation of results Results are presented in a clear way with the table provided.However the authors could make a graph with the bacteria identified as a percentage of samples to help readers.

3 .
How the style and organization of the paper communicates and represents key findings Key findings are clearly presented 4. Literature analysis or discussion Certain points can be added to the discussion (see Major points 1, 2)Please rate the manuscript for methodological rigour GoodPlease rate the quality of the presentation and structure of the manuscript Very goodTo what extent are the conclusions supported by the data?Partially supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes SciScore report https://doi.org/10.1099/acmi.0.000612.v1.1 © 2023 The Authors.This is an open-access article report distributed under the terms of the Creative Commons License.

Table 1 .
Clinical and laboratory characteristics The manuscript is describing an application of multiplex PCR in pleural effusion.It is interesting and well written.Overall, the methodology is sound, the results well presented and the literature sources of good quality and relevance.Few minor corrections: Abstract: provide values and statistics Abstract: At the end provide a sentence about the medical importance of your findings.Methods: provide more details about the statistical tests that were used.The Discussion needs more references of similar studies.Are you sure there are only two?What about non-empyema cases?All microbial genus names have to be in italics In this manuscript by London-Ruiz et al, the authors evaluate a multiplex-pcr platform to identify the etiological agents of pleural effusion in pediatric pneumonia.They use this platform on 17 cases identifying the agent and compare this method with the standard procedure of bacterial cultures.The authors reach the conclusion that this method is worthy to be considered for implementation in clinical diagnosis.Although the results presented in the table are clear, there are a few things that should be clarified before the manuscript is suitable for publication.Major points: 1.